Process of extracting chloroplasts



o '45 the extraction plant I of supply, and other features influence thechoice Patented Mar. 23, 1937 UNITED STATE s PATENT OFFlCE;

PROCESS OF EXTBACTIlG OHLOBOPLASTS Robert H. Van Sant, Detroit, Mich,.mesne assignments, to American MIM by Chlorophyll,

me, a corporation of Delaware No Drawing. Application June a, 19::Serial 50mm. rotor-ea) and xanthophyll. The first two of these areclosely related and for some purposes may be considered as one. They areusually extracted as one and separated afterwards, though the contraryis possible. Prior investigations have re-. sulted in the conclusionthat these four pigments are to be found identical in all plants,andcomparative investigation has indicated that chlorophyll'is ofconstant composition in all appearances. v All heretofore publishedmethods of extracting chlorophyll from plants, while based on a suitablesolvent ,for extracting the chlorophyll, the action of the solvent hasbeen greatly retarded due to the fact, that all plant material containsconsiderable starchy material. In most cases incomplete extractionhasresulted.

In the method here disclosed the sugars and they are washed out withwater. Then the chlorophyll solvent can penetrate more readilyandcompletely, whereby the extraction is expedited and the yield increased.

Chlorophyll isthought to contain 4.5% of magnesium in each molecule. IWillstatter is quotedas saying that fresh leaves (without identifyingthe leaves of what vegetable) contain about two parts oichlorophyll (G)in a thousand, three-fourths (b) and one-thirdoi a part of xanthophylland one-sixth 'of a part of carotin. 9 By proceeding according to thisinvention, ex-

tractions as high as nine-tenths per cent chlorophyll have beenobtained, or nine parts in athou sand;

The choice of vegetable matter, the location of with respect to thdsouwgof the preliminary treatment.- According to the preferred procedure fora plant locatedin a northern climate, dry leaves will be the basis ofoperation and, generally speaking, these may be taken from any greenvegetation Among those now known to be'suitable may be listed stingingnettles, mulberry, spinach, alfalfa, clover, pine 55 needles, but theseare suggestive only.

. fineness. The

- soluble in some solvent starch is treated with a digestant te -convertit into soluble of the comminuted leaves in a of a part of chlorophyllAir drying the shade appears to give very good results, perhaps becauseit is done at temperatures which do not damage the pigments,-andthere-is no bleaching action, etc., whereas assuming the supply isremote from the place of extraction, the cost of shipping, the danger ofinvJul-y en route, etc. are eliminated by drying in the shade at or nearthe place of growth and gathering.

The steps of the process may, for convenience of description, beindicated numerically as 011 lows:

(1') Comminute the dry leaves to a suitable means and mariner ofaccomplishing this are a matter of wide selection, the principal careshould be to avoid high temperatures, or any great range from temperateatmosphere. i

(2) Wash'the comminuted leaves with ether to remove. waxes'and oils.This is particularly desirable in case do! vegetation of oily ;,or waxycharacter.

(3) Reduce dition' or character in the starch components to a coniniurethe pigment.

water about eight andone-half times the volume suitable vesselproximately correch equipped to maintazin a temperature within 2'. plusor minus of 98 Add one gram of amylacecus-enzymes (such as I amylopsln,etc.) .tested and proven, one to two hundred; that isflone gram vwillconvert two hundred grains of starch into sugar in less thanfifteenminutes at 98.2 r. a a

Alter the amylaceous enzymes have been thor- O hly stirred into thewater, add the comminuted leaves and agitate thoroughly. until allstarch is. converted into sugar. (The time recan be determined bytesting samples. One to four hours has proven sufllcient.)

(4) Wash out the soluble components with The accomplishment of thisindicated when the water runs clear and is without odor:

' 5) Macerate the residue (which may be called mare, and includeschiefly the pigments mentioned, with lignin and cellulose material) witha solvent selected according tothe product sought. Ethyl alcohol serveswell when the extract is to be treated with hydrochloric acid and otherinwhich they are-readily that will not dissolve or step will be organicacids to produce pheophytin. Acetone is suitable when magnesiumchlorophyll is to be the Product.

About 1700 c. c. of acetone will be satisfactory B for the firstmaceration, to be followed by-others with more or less acetone,according to thecondition. The step is complete when the solvent drawnoff shows no trace of color.

(6) Distill, evaporate, or otherwise recover the 10 solvents and collectthe residue, which will consist chiefly of the pigments if the above hasbeen followed.

Processes for refinement, conversion and application to science andindustry are known and reference to the work of Willstatter and Stoll,translated by Scherz and Mertz, will suifice.

The foregoing has been found particularly satisfactory with driedmulberry leaves and alfalfa shipped green from the place of picking, anddried stinging nettle leaves of commerce purchased in the open market.Suitable variations will have to be made for other basic materials whichare so various that an attempt to indicate appropriate variations foreach would be useless.

Green leaves may be comminuted without dryingout but, of course, theyare more difficult to handle in a mill, and dried leaves can be muchmore cheaply shipped from the place of gathering to the extractionplant.

Amylaceous enzymes mentioned in step (3) may be replaced, orsupplemented with an extract of the salivaryglands or ruminants, oranimal diastase, or malt diastase or, in general, any treatment thatwill make the starch soluble in water without damaging the pigments. I

At this point it is well to mention that the enzymes in the digestivetracts of fowls or fish will be found to conform to the principle ofthis methd of extraction. In the case of fish the mass 40 may be treatedat a much lower temperature. The water used should conform to theconditions of its habitat. The important thing in-this process is towork in the proper temperatures previously determined for theigreatestpotency of the 45 individual digestant used.

The fineness of the grinding or milling will vary with the leaves andsome out and try will be necessary to determine the optimum for aparticular material. With" mulberry leaves picked 50 in Florida in earlyspring 40 mesh gave excellent results, while alfalfa picked in Ohio inMay worked better at 20 mesh. Dog fennel leaves have very littlecellulose and may be treated with little or no grinding. 5 I claim as myinvention:

1. The process of extracting chloroplasts from starch-containingvegetation whichcomprises comminuting leaves containing chloroplasts,mixing the comminuted mass with water at approxi- 0 mately bloodtemperature, adding amylaceous enzymes for converting the starch of saidenzymes into sugar, washing out the soluble matter with water,macerating the residue, treating said real-- due with a chloroplastsolvent,, removing the liquid content from the mass, and finally evapo-I rating the solvent from said liquid content.

2. The process of removing chloroplasts from starch-containing vegetablematter which contains chloroplasts comprising comminuting said vegetablematter, converting the starch into sugar by treating the mass with anamylaceous enzyme while said mass is in a warm condition but below atemperature that would destroy said enzyme, washing out the solublematter from the mass .with water, removing the'liquid from the mass,removing the chloroplasts by treating the residue with a chloroplastsolvent of the class consisting of ethyl alcohol and acetone, andfinally removing the solvent from the liquid by evaporation.

3. The process of removing chloroplasts from starch-containing vegetablematter which contains chloroplasts comprising comminuting said vegetablematter, removing the waxes and oils by treating the comminuted mass withether, converting the starch into sugar by treating the residue with anamylaceous enzyme while said residue is in a warm condition but below atemperature that would destroy said enzyme, washing out the solublematter from the mass with water,

removing the liquid from the mass, removing the chloroplasts by treatingthe residue with a chloroplastsolvent of the class consisting of ethylalcohol and acetone, and finally removing the solvent from the liquid byevaporation.

4. The process of removing chloroplasts from starch-containing vegetablematter which contains chloroplasts comprising comminuting said vegetablematter, converting the starch into sugar by, treating the mass with anamylaceous enzyme while said mass is in a warm condition but below atemperature that would destroy said enzyme, washing out the solublematter from the mass with water, removing the liquid from the mass,removing the chloroplasts by treating the residue with acetone, andfinally removing the acetone from the liquid by evaporation.

5. The process of removing chloroplasts from starch-containing vegetablematter which contains chloroplasts comprising comminuting said vegetablematter, converting the starch into sugar by treating the mass with anamylaceous enzyme while said mass is at a temperature within 2 F. aboveor below 982 F., washing out the soluble matter from the mass withwater, removing the liquid from the mass, removing the chloroplasts bytreating the residue with a chloroplast solvent of the class consistingof ethyl alcohol and acetone, and finally removing the solvent from theliquid by evaporation.

ROBERT H. VAN SANT.

